The present invention pertains, in general, to the production of seedlings which demonstrate better emergence characteristics and improved seedling growth when grown under low light conditions. In particular, the present invention pertains to modifying the genotypes of plant cells to include a sequence coding for the Coil domain of the COP1 protein.
All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
Through photosynthesis, light provides the energy source for plants and, ultimately, for all living organisms. The light environment plays a crucial role in plant growth and development. Besides serving as a source of energy, light provides signals to regulate many complex developmental processes. At least three photoreceptor familiesxe2x80x94pytochromes (red and far-red light), blue light receptors, and UV light receptorsxe2x80x94mediate these light-regulated developmental processes. Light signals perceived by specific photoreceptors are transduced via signaling components to bring about the diverse downstream physiological responses, including seed germination, stem elongation, chloroplast and leaf development, floral induction, and coordinated expression of many light-regulated nuclear- and chloroplast-encoded genes.
In response to a fluctuating environment, the nonmobile plant must be able to sense varying light signals and to optimize growth and development. Higher plants possess sophisticated photosensory and signal transduction systems to monitor the direction, quantity, and quality of the light signal and to adjust their growth and development through regulated gene expression at every stage of their life cycle, such as germination, seedling development, and flowering. These light-regulated developmental processes are collectively termed photomorphogenesis.
Plant development is a highly malleable process that is strongly influenced by environmental factors, especially light. The effects of light on plant development are especially prominent at the seedling stage (Kendrick and Kronenberg, 1994; McNellis and Deng, 1995). As compared with plants grown in light, those grown in darkness are white or yellow in color, the internodes are long, the leaves are very much reduced in size, and the root systems are poorly developed. This condition is known as etiolation. Of course, etiolated seedlings cease growth when their reserve food supply is exhausted. While the effect is less dramatic than when plants are grown in darkness, the etiolation effect also occurs under low light levels as well.
The light environment in nature is complex. Unobstructed sunlight consists of a wide continuum of photon wavelengths that is conveniently divided into three large spectral domains: UV ( less than 400 nm), visible (400 to 700 nm) and far-red ( greater than 700 nm) light. The spectral quality, or relative photon distribution, at different wavelengths can vary greatly, depending on the location and the time of day. For example, within the canopy, the light available is essentially depleted in the visible and UV regions, and far-red light is highly represented. Furthermore, twilight normally has a higher far-red to red ratio than daylight. Although higher plants effectively utilize only visible light for photosynthesis, they have the capability to sense and respond to a much wider range of the spectrum, including UV and far-red light.
In a photochemical process such as photosynthesis, the end product depends upon the number of quanta absorbed rather than the total light energy absorbed. A single red photon has the same effect in photosynthesis as a single blue photon, for example, although the blue photon has more energy. Hence, in the recent literature it has become common to refer to the number of photons per unit area per unit time. Einsteins (for photosynthesis) or microEinsteins (for low light responses) are used. While an open field during a mid-summer day may receive as much as 2,000 microEinsteins per square meter per second, the same area in an indoor room with fluorescent lamps may only receive 50 to 100 microEinsteins per square meter per second. When the open field has its light blocked by smog, clouds or rain, it may actually register less photons per unit area per unit time than the indoor room.
In general, absence of light increases, and presence of light decreases, the rate at which the stems elongate. Thus, the features associated with etiolation ensure, under natural conditions, that the shoot is carried towards the light as rapidly as possible. Such a physiological and morphological response to the complete lack of light is critical for the growth and eventual emergence of the seedling from the position where the seed is planted in the soil or other growth media.
The etiolation process does not necessarily cease once the seedlings successfully emerge from the soil or other growth media. In many plants, there is a rhythmic night-and-day growth rate of the shootxe2x80x94greater at night than during the day, provided that the temperature at night does not fall too low. Plants grown in full light have shorter and sturdier stems and somewhat thicker leaves than those grown in the shade. One result of crowding plants is a reduction in the light intensity to which they are exposed. In an effort to reach higher light levels, the shaded plants develop longer and more spindly stems than those grown under less crowded conditions. Thus, in some circumstances, the etiolation process can be viewed as a survival tactic.
However, under some conditions of low natural light, such as would result from a succession of smoggy, cloudy or rainy days or from an inability to supply high levels of artificial light in the greenhouse, etiolation can cause plant husbandry problems. For example, etiolated seedlings tend to fall over easily and to produce weaker plants which are more susceptible to pests, such as aphids and spider mites, and to other environmental challenges, such as wind or water-logged pots or fields. Under such conditions, the etiolated seedlings may develop into less vigorous adult plants, produce less reproductive structures and fewer offspring, or even perish. If a sufficient number of seedlings or plants are adversely affected by the etiolation effect, this may result in reduced production of a particular plant product on a per surface area yield basis. For example, the yield of tomato fruits on a per hectare or per acre basis may be dramatically reduced if severe seedling etiolation results in the formation of spindly tomato plants which lodge. Such lodging can reduce fruit size through decreased photosynthetic activity of the collapsed shoot, greatly increase fruit rotting through contact of the fruit with the soil, and lead to greater pest access and pest damage of the fruits. While the spindly tomato plants could be supported on trellises, stakes or cages, such rescue attempts are very labor intensive and can be prohibitively costly for large-production operations. In addition, such increased xe2x80x9cman-handlingxe2x80x9d of the plants can also result in further plant breakage and fruit droppage.
Plant responses to light are especially evident in the young seedling, although they occur throughout the life of the plant. Early seedling development in Arabidopsis (Arabidopsis thaliana) provides an excellent model system to dissect the light signal transduction pathway in plants. As a typical dicotyledonous plant, Arabidopsis seedlings follow two distinct strategies of development, skotomorphogenesis in darkness or photomorphogenesis in light. Thus, Arabidopsis seedlings display contrasting developmental patterns in the presence or absence of light. In selecting for and characterizing late germinating mutants of Arabidopsis, Michelle Stopper and James J. Campanella (Arabidopsis thaliana Database, http://genome.www.stanford.edu/Arabidopsis) used high-intensity light conditions of approximately 450 microEinsteins per square meter per second and low-intensity light conditions of approximately 100 microEinsteins per square meter per second.
Under normal light conditions, seedlings follow photomorphogenic development characterized by inhibition of hypocotyl elongation, development of expanded cotyledons, biogenesis of chloroplasts, and expression of light-inducible genes.
In darkness, seedlings etiolate, displaying elongated hypocotyls, closed and unexpanded cotyledons, and apical hooks. Also, the light-inducible genes are repressed, and plastids develop into non-photosynthetic etioplasts in the darkness. This developmental commitment is plastic and reversible; the etiolated seedlings can dynamically respond to incoming light stimuli and initiate photomorphogenesis (for review see Chory 1993; McNellis and Deng, 1995).
The typical physiological and morphological response to growing in the dark (i.e., elongated hypocotyls, closed and unexpanded cotyledons and apical hooks) produces seedlings which are better able grow through the soil and penetrate the soil surface. Such natural seedling growth is even more important for seedlings which germinate from seeds planted at greater depths than normal. If the cotyledons were to open and expand under the soil, the increased soil resistance would impede further upward growth, requiring the seedlings to expend greater energy for continued upward growth. Such premature cotyledon expansion and increased physical resistance could result in damaged cotyledons and possibly an exhaustion of food reserves prior to seedling emergence.
As discussed above, photomorphogenic development depends on the plant being able to detect light signals. At least three families of photoreceptors, phytochromes (red and far red light), blue light receptors, and ultraviolet (UV) light receptors, are utilized to sense the different light wavelengths, and the signals transduced by these receptors coordinately regulate the transcription of specific genes.
It is not fully understood how light stimuli perceived by multiple photoreceptors are transduced and integrated to affect developmental programs. If the ability of a plant to detect light signals is impaired, such as when photoreceptors are disrupted by mutations, then a growing seedling assumes a somewhat etiolated developmental pattern. Genetic screens of Arabidopsis seedlings, based on either etiolated phenotypes under light conditions or photomorphogenic phenotypes in complete darkness, have identified a large number of the light-signal transduction components involved in controlling seedling development (for review see McNellis and Deng, 1995).
A first class of mutant seedlings with reduced light-responsiveness display characteristic long hypocotyl (hy) phenotypes. This class of mutants defines positive regulators of photomorphogenesis including photoreceptors (e.g. phyA, phyB, and hy4) and components acting downstream of specific photoreceptor (e.g. fhy1,fhy3, and red1) or multiple photoreceptors (e.g. hy5). (Chory 1992; Whitelam et al., 1993; Wagner et al., 1997). For example, mutations in phytochrome A gene (PHYA) cause reduced responsiveness to far-red light signals (Dehesh et al., 1993; Nagatani et al., 1993; Parks and Quail, 1993; Whitelam et al., 1993); mutations in phytochrome B gene (PHYB) cause reduced responsiveness to red light stimulation (Reed et al., 1993; Wester et al., 1994); mutations in a putative blue light receptor gene, HY4, cause reduced responsiveness to blue light (Ahmad and Cashmore, 1993). Symptoms of reduced light responsiveness in phyA, phyB, and hy4 mutants include long hypocotyls and reduced cotyledon expansion under certain light conditions (Koornneef et al., 1980; McNellis et al., 1994b). On the other hand, photoreceptor overexpression causes hypersensitivity to the spectral quality of light that the photoreceptor primarily absorbs. For example, overexpression of PHYB causes hypersensitivity to red light (Wagner et al., 1991; McCormac et al, 1993); overexpression of PHYA causes hypersensitivity to far-red light and red light (Boylan and Quail, 1991; McCormac et al., 1991, 1992, 1993; Whilelam et al., 1992); overexpression of the CRYPTOCHROME1 (CRY1) blue light photoreceptor encoded by the HY4 locus causes hypersensitivity to blue, UV-A, and green light (Lin et al., 1995). Because of the importance of light to plant survival, it makes sense that plants have developed multiple photoreceptors with partially overlapping functions. The photoreceptors work together to monitor light signals, and stimulation of any one photoreceptor class alone appears to be sufficient to induce many aspects of seedling photomorphogenesis (Kendrick and Kronenberg, 1994; McNellis and Deng, 1995).
Several excellent reviews deal more extensively with the photoreceptor phytochromes (Quail, 1991; Furuya, 1993; Viestra, 1993), with light regulation of gene expression (Gilmartin et al, 1990; Thompson and White, 1991; Kaufman, 1993) and with photomorphogenic mutations (Chory, 1993).
The recent molecular identification of HY5 as a bZIP transcription factor may provide a tool to bridge the light signal transduction pathway to the control of gene expression (Oyama et al., 1997).
The second class of mutants includes those that display constitutive photomorphogenesis, namely constitutive photomorphogenic (cop), de-etiolated (det) and fusca (fus) mutants (reviewed by Wei and Deng, 1996). Genetic studies indicate that their gene products function as negative regulators acting downstream of multiple photoreceptors, including phyA, phyB, and the blue-light receptor CRY1 (McNellis and Deng, 1995). While a subset of these mutants are implicated in playing a role in phytohormone signaling (Chory and Li, 1997; Kraepiel and Miginiac, 1997), ten of the pleiotropic and essential COP/DET/FUS loci are believed to be responsible for mediating the suppression of photomorphogenic seedling development in darkness (Wei and Deng, 1996).
The pleiotropic CONSTITUTIVE PHOTOMORPHOGENIC/DEETIOLATED/FUSCA (COP/DET/FUS) loci may define a group of important developmental regulators specifically involved in light control of seedling morphogenesis. Mutations at all of these loci cause seedlings to exhibit essentially all aspects of photomorphogenic development in darkness (Castle and Meinke, 1994; Misxc3xa9ra et al., 1994; Pepper et al., 1994; Wei et al., 1994; Kwok et al., 1996). Because all of the mutations at the pleiotropic COP/DET/FUS loci are recessive and cause constitutive photomorphogenic development regardless of the actual light conditions, the proteins encoded by these loci have been postulated to act as repressors of photomorphogenesis in the dark. Light signals absorbed by the various photoreceptors are thought to reverse the repressive activities of the COP/DET/FUS proteins and allow photomorphogenic development to proceed (for a recent review, see McNellis and Deng, 1995).
Putative null mutations at all the COP/DET/FUS loci cause adult lethality and severe physiological abnormalities during seed maturation and seedling development. This has raised concern regarding the specificity of these genes in regulating light-mediated development (Misxc3xa9ra et al., 1994; Castle and Meinke, 1994). It is formally possible that the pleiotropic COP/DET/FUS proteins may be ubiquitous global cellular regulators and function mainly to set up the cellular environment necessary for light regulatory cascade. Although this alternative model is also consistent with the mutant phenotypes of those genes, it suggests that their gene products are not an integral part of the light regulatory cascade.
The molecular cloning of four pleiotropic COP/DET/FUS genes, namely COP1, COP9, DET1, and FUS6 (COP11) provides an opportunity to understand the molecular mechanisms of repression of photomorphogenesis (Deng et al., 1992; Castle and Meinke, 1994; Pepper et al., 1994; Wei et al., 1994). COP9, DET1, and FUS6 encode novel xcex1-helical rich proteins that constitutively localize in nucleus (Pepper et al., 1994; Wei et al., 1994; Chamovitz et al., 1996; Staub et al., 1996). COP9 has been found to be a part of an eight subunit protein complex consisting of COP9, FUS6 (COP11), presumably COP8, and others (Wei et al., 1994; Chamovitz et al., 1996; Wei and Deng, 1996 and 1998; Wei et al., 1998). COP1, on the other hand, appears to function as an autonomous repressor of photomorphogenesis based on previous experiments in modulating COP1 cellular activity. For instance, overexpression of full-length COP1 causes reduced light responsiveness (McNellis et al., 1994b), while overexpression of a dominant negative mutant form of COP1 results in hypersensitivity to the light and a partial de-etiolation in darkness (McNellis et al., 1996). Cell biological studies using a fusion protein of COP1 with a reporter xcex2-glucuronidase (GUS) protein revealed the light regulated nucleocytoplasmic partitioning of COP1 may be one of the mechanisms of how light negatively regulates the repressors of photomorphogenesis (von Arnim and Deng, 1994).
Moderate overexpression of COP1 in Arabidopsis has been shown to partially suppress blue and far-red light-mediated inhibition of hypocotyl elongation and blue light-mediated cotyledon expansion (McNellis et al., 1994b). Because those effects are the only phenotype that can be detected in those transgenic plants, it was, therefore, interpreted as evidence supporting COP1""s direct involvement in the light signaling cascade. However, overexpression of full-length COP1 failed to have any detectable effect on the phytochrome B-mediated red light inhibition of hypocotyl elongation (McNellis et al., 1994b), possibly due to the low levels of overexpression. In addition, if COP1 functions directly within the light regulatory cascade and acts as a light-inactivable repressor of photomorphogenic development, ideally COP1 overexpression should also affect light control of plastid development and light-regulated gene expression. Limited overexpression of the COP1 protein (four fold or less) is clearly unable to cause detectable effect on the light-regulated gene expression and plastid development (McNellis et al., 1994b). Therefore the previous overexpression studies could not critically rule out the possibility that COP1 overexpression coincidentally influenced cell elongation in the hypocotyl and cell expansion in the cotyledon under our experimental conditions through a mechanism unrelated to photomorphogenesis.
To overcome the limitations of the full-length COP1 overexpression studies, we initiated an effort to overexpress specific mutated forms of COP1 to look for possible dominant-negative effects. COP1 is a 76.2 kD protein with an N-terminal Ring-finger zinc-binding domain, followed by a putative xcex1-helical domain, and multiple WD-40 repeats in the C-terminal half that are similar to the xcex2 subunit of trimeric G-proteins (Deng et al., 1992; McNellis et al., 1994a). The complete nucleotide and amino acid sequence of COP1 was previously provided by Deng et al. (1992b) and is available as NCBI Accession Number L24437.
Thus, COP1 encodes a protein with a novel combination of three structurally recognized domains, namely an N-terminal Zn-binding domain (hereinafter called the Ring-finger domain), a putative coiled-coil region (hereinafter referred to as the Coil or coiled-coil domain), and C-terminal WD-40 repeats (Deng et al., 1992; McNellis et al., 1994a). The 282 amino acid N-terminal fragment of COP1, including the Ring-finger and Coil domains, is referred to as xe2x80x98N282xe2x80x99.
The Ring-finger domain comprises eight metal ligands with a consensus of C3HC4 and binds two zinc atoms in a unique tetrahedral xe2x80x98cross-bracexe2x80x99, thus forming one integrated structural unit (von Arnim and Deng, 1993; for review see Berg and Shi, 1996; Borden and Freemont, 1996; Saurin et al., 1996).
The Coil region is predicted to be an xcex1-helical structure capable of forming a superhelix (Lupas, 1996).
The WD-40 motif is about 40 amino acids in length and contains a highly conserved tryptophan-aspartate (WD) sequence (for review see Neer et al., 1994). Proteins with Ring-finger or WD repeats are involved in a wide variety of processes, including gene repression, oncogenesis, and signal transduction (for review see Neer et al., 1994; Borden and Freemont, 1996; Saurin et al., 1996). Studies in other systems have implicated roles in protein-protein interactions for all three modules, suggesting that the pleiotropic role of COP1 may be achieved through interactions with multiple proteins. However, the specific functional roles of the COP1 modules have not been addressed.
To understand the structural implications of these structural motifs, recessive mutations of the COP1 gene were isolated based on their constitutive photomorphogenic seedling development in darkness (McNellis et al., 1994a). All of the mutant lines produced dark-grown seedlings that mimicked wild-type seedlings grown in the light. The phenotype of the dark-grown mutant seedlings included: short hypocotyls, open and enlarged cotyledons, accumulation of anthocyanin, cell-type differentiation and chloroplast-like plastid differentiation in chloroplasts. Moreover, in more prolonged dark-growth periods the mutants exhibit true leaf development that parallels that in light-grown seedlings. The four mutant alleles represent two types of mutations: three alleles (cop1-1, cop1-2 and cop1-3) have severely affected phenotypes whereas one allele (cop1-4) has a less severe phenotype. Compared to the severe alleles, the cop1-4 mutant has slightly longer hypocotyls in dark-grown seedlings and does not accumulate abnormal levels of anthocyanin. Adult light-grown plants homozygous for cop1 mutations are much smaller than wild-type (rosette diameter of about 0.5-1 cm, compared to about 5-10 cm for wild-type plants) and have much lower fertility than wild-type plants when grown under the same conditions (Deng and Quail, 1992).
The cop1-4 mutant allele produced a COP1 protein with only the N-terminal 282 amino acids, including both the zinc binding and the coiled-coil domains. The weak cop1-4 allele represents a C-to-T mutation that changes the Gln-283 CAA codon to a UAA stop codon. No wildtype size COP1 protein has been observed in the protein gel blot analysis of cop1-4 mutants, suggesting that translation through the newly created stop codon, if any, is negligible (McNellis et al., 1994a). The cop1-4 mutants are still capable of reacting to light to a certain degree. Dark-grown seedlings of cop1-4 develop short hypocotyls and expanded cotyledons (Ang and Deng, 1994). However, although the allele only produces a weak phenotypic defect in the seedling stage, it causes severe size reduction of light-grown plants and greatly reduced seed set (Deng and Quail, 1992).
Previously we reported COP1 overexpression studies using stably transformed Arabidopsis plants containing transgenes encoding the N-terminal half of COP1 , which contains both the Ring-finger domain (the Zn-binding motif) and the Coil domain (the putative coiled-coil domain), but depleted of the C-terminal half of COP1, which contains the entire WD-40 repeat domain (McNellis et al., 1996). The transformed plants contained DNA coding for the full-length COP1 protein as well as additional, separate DNA which encoded the N282 fragment. Overexpression of the N-terminal 282 amino acid fragment (N282) of COP1 caused a dominant negative interference with the ability of the endogenous wildtype COP1 to suppress multiple photomorphogenic development processes, including cellular differentiation, plastid development, and gene expression. This effect of N282 overexpression seems to be specific for light-regulated development, because it has no effect on stress- and pathogen-inducible gene expression. Furthermore, the phenotype of all of the transgenic N282 lines is limited to the light control of seedling development, and little effect was detected on other developmental processes and normal adult development.
As discussed above, high-level expression of the N282 fragment caused a dominant-negative phenotype similar to that of the loss-of-function cop1 mutants. The phenotypic characteristics include hypersensitivity of hypocotyl elongation to inhibition by white, blue, red, and far-red light stimuli. In the dark, N282 expression led to pleiotropic photomorphogenic cotyledon development, including cellular differentiation, plastid development, and gene expression, although it had no significant effect on hypocotyl elongation. The results implied that the N282 COP1 fragment, which contains the Ring-finger and Coil domains, is capable of interacting with either downstream targets or with the endogenous wild-type COP1 , thus interfering with normal regulatory processes. We concluded that N282 of COP1 are involved in protein-protein or protein-nucleic acid interactions that are essential for the light control of seedling development by COP1 (McNellis et al., 1996).
Work conducted using the cop1 gene of Arabidopsis has a direct bearing on the seedling growth of other plants, including plants of agronomic and horticultural importance. For example, Frances et al. (1992) have noted that a pea (Pisum sativum) mutant with light-independent photomorphogenesis, designated lip1, had several features in common with the deetiolated Arabidopsis mutants det1, det2 and cop1. However, the researchers also noted several important differences, including varying effects on phytochrome levels, organ-specific gene expression, plastid development and response to dark adaptation. Zhao et al. (1998) cloned and sequenced the cop1 gene from pea (NCBI Accession Number Y09579). Sequence comparison between Cop1 proteins of pea and Arabidopsis revealed that the two Cop1 proteins were highly homologous in the regions with functional domains and at the C-terminus. The two Cop1 proteins are 77.5% identical and 86.9% similar in amino acid sequences. Since pea plants display typical photomorphogenesis of the higher plants, this result demonstrates that the findings in Arabidopsis have a direct bearing on the higher plants of economic importance.
McNellis et al. (1994a) noted that the C-terminal COP1 sequence had a high degree of conservation with corresponding regions of COP1 homologs in spinach (Spinacia olereacea) and rice (Oryza saliva). The observation regarding rice was particularly interesting since the morphogenic development of monocotyledonous plants is different from that of dicotyledonous plants. For example, unlike dicotyledonous plants such as Arabidopsis and spinach, monocotyledonous plants such as rice exhibit extensive leaf development in darkness. Tsuge et al. (1997) showed that a COP1 homologue exists as a single copy gene in the rice genome and that the three structural domains are highly conserved between Arabidopsis and rice.
A homologue of the Arabidopsis COP1 gene has also been cloned from tomato (TCOP1) (Frances et al., 1997). The deduced amino acid sequence of the tomato gene shows high identity to the Arabidopsis gene particularly in the three defined structural motifs.
As discussed above, there exists a need for seedlings which demonstrate improved growth characteristics under low light conditions. While seedlings of the cop1-4 mutant of Arabidopsis display somewhat shorter hypocotyls when grown under low light conditions as compared to wildtype seedlings grown under the same conditions, these mutant seedlings become unhealthy, non-productive adult plants. These mutants fail to produce any COP1 protein.
Wildtype plants transformed with the N282 fragment of COP1 display a seedling phenotype when grown under low-light conditions which is characterized by shorter hypocotyls when compared to wildtype seedlings grown under the same low light conditions (McNellis et al., 1996). However, as detailed herein and by McNellis et al. (1996), N282-transformed plants have the distinct disadvantage of also displaying abnormal cotyledon growth and expansion when grown in the dark. This aspect of the N282-transformed plants would have detrimental effects on upward seedling growth and seedling emergence, as discussed previously. Thus, there remains a need for seedlings which display normal or near-normal emergence characteristics under dark conditions and reduced seedling etiolation under low light conditions. Prior to this invention, such plants had not been realized.
This invention involves the functional dissection of COP1 domains, by utilizing a combination of reverse-genetic, biochemical, and cell biological approaches. Our discovery reveals the distinct but overlapping functions of COP1 domains in the light control of seedling development and provides a structural basis for COP1 as a molecular switch.
Our data suggests that COP1 acts primarily as a homodimer, and likely dimerizes through the coiled-coil domain. The present invention relies on the unexpected discovery that the Ring-finger and the coiled-coil domains can function independently as light-responsive modules mediating the light controlled nucleocytoplasmic partitioning of COP1. The C-terminal WD-40 domain functions as an autonomous repressor module since the overexpression of COP1 mutant proteins with intact WD-40 repeats are able to suppress photomorphogenic development. This WD-40 domain-mediated repression can be at least in part accounted for by COP1""s direct interaction with and negative regulation of HY5, a bZIP transcription factor that positively regulates photomorphogenesis. However, COP1 self-association is a prerequisite for the observed interaction of the COP1 WD-40 repeats with HY5. This work thus provides a structural basis of COP1 as a molecular switch.
Using the discovery of this invention, one skilled in the art can produce plants which demonstrate better emergence characteristics combined with improved seedling growth under low-light conditions.
This invention comprises methods of altering the emergence of seedlings under extremely low light or darkness conditions and the subsequent growth of the seedlings under low light conditions. More specifically, this invention provides plants with the following physiological and morphological characteristics: 1) the seedling phenotype displayed by the plants under extremely low light conditions or in complete darkness is characterized by a lack of leaf expansion similar to that of wildtype seedlings under the same conditions; and 2) the seedling phenotype displayed by the plants under low light conditions is characterized by shorter hypocotyls when compared to wildtype seedlings grown under the same low light conditions. Even more specifically, this invention is directed to altering the emergence characteristics of seedlings in the dark and the subsequent post-emergent growth of seedlings under low light conditions by introducing a nucleotide sequence coding for the Coil domain amino acids of the COP1 gene.
This invention provides plant cells which comprise a COP1 gene and, separately, a nucleotide sequence coding for the Coil domain the COP1 gene. Thus, the plant cells provided by this invention produce both the Coil domain protein and the wildtype COP1 protein. This invention also provides plants produced from the plant cells of this invention.
The invention also encompasses transforming plants with a nucleotide sequence for COP1 wherein the WD-40 and Ring-finger domains have been disabled. There are a number of ways in which the Ring-finger and WD40 repeats can be effectively disabled in a manner that will reduce or eliminate their expressivity and function. Thus, this invention provides plant cells comprising a COP1 gene and, separately, a nucleotide sequence coding for the Coil domain of the COP1 gene, wherein the plant cell does not have a separate expressible nucleotide sequence coding for either the Ring-finger domain of the COP1 gene or for the WD-40 domain of the COP1 gene associated with said Coil domain nucleotide sequence.
This invention provides a plant comprising a nucleotide sequence coding for the Coil domain of the COP1 gene, wherein the seedling phenotype displayed by the plant during emergence is characterized by unopened leaves and subsequent seedling growth under low light conditions after emergence is characterized by shorter hypocotyls when compared to wildtype seedlings grown under the same low light conditions. The adult plant produced by the methods of this invention have approximately the same shoot size and seed set as the wildtype adult plant grown under the same conditions.
This invention also provides isolated DNA molecules encoding the Coil domain of COP1.
This invention further provides vectors comprising isolated DNA molecules encoding the Coil domain of COP1. This invention further provides such vectors which also include a promoter operably linked to the isolated DNA sequence.
This invention also provides host cells transformed with such vectors, wherein the host cells are prokaryotic cells, fungal cells or photosynthetic eukaryotic cells. Thus, this invention provides transgenic procaryotic, fungal or photosynthetic eukaryotic organism wherein the DNA sequence coding for the Coil domain protein is incorporated into the genomic DNA of the organism thereby producing transgenic organisms which produce Coil domain protein. More specifically, the transformed photosynthetic eukaryotic host cells provided by this invention include both monocotyledonous and dicotyledonous plants. Even more specifically, the transformed monocotyledonous and dicotyledonous plants provided by this invention include Arabidopsis, spinach, tomato, pea and rice.
This invention includes plants regenerated from the transformed plant cells as well as the progeny of such transformed plants, wherein the progeny also produce Coil domain protein.
This invention further provides methods of modifying the normal function of the endogenous wildtype COP1 gene, wherein the methods comprise: 1) preparing vectors comprising an isolated DNA sequence encoding the amino acids for the Coil domain of COP1; and 2) inserting the vectors into cells selected from the group consisting of prokaryotic cells, fungal cells and photosynthetic eukaryotic cells to produce transformed cells. The methods of this invention further provide for regenerating transformed plants from the transformed photosynthetic eukaryotic cells. The methods of this invention even further provide for the production of transformed progeny from the transformed regenerated plants. Furthermore, this invention further provides methods of sexually crossing the regenerated transformed plants with other plants; harvesting the resultant seed; growing seedlings from the resultant seed under extremely low light levels and selecting those that have little or no leaf expansion; and growing the selected seedlings from step c under low light levels and selecting transformed seedlings which have shorter hypocotyls than wildtype seedlings grown under the same low light conditions.
This invention also provides methods of producing transformed seedlings which have modified phenotypes during seedling emergence in darkness and when grown as seedlings under low light conditions, wherein the methods comprise: 1) preparing vectors comprising an isolated DNA sequence encoding the amino acids for the Coil domain of COP1; 2) inserting the vectors into plant cells; 3) producing viable transformed parental plants from the transformed plant cells; and 4) growing transformed seedlings from the seed produced on the viable transformed parental plants. Following the procedures outlined herein, one of ordinary skill in the plant breeding art can produce modified seedling phenotypes characterized by transformed seedlings having substantially unopened, compact, wildtype leaves during seedling emergence in the darkness and reduced etiolation of transformed seedlings under low-light levels when compared to non-transformed, wildtype seedlings grown under the same low light conditions. The methods of this invention also provide modified seedling phenotypes characterized by transformed seedlings having substantially wildtype leaf development during seedling emergence in the darkness and shorter, more vigorous stems and greener, more developed leaves of transformed seedlings when compared to non-transformed, wildtype seedlings grown under the same low light condition.
This invention also provides methods of growing crops wherein the crop includes one or more plants which contain a DNA sequence coding for the Coil domain protein and which also contain the indigenous wildtype COP1 gene. The plants with the Coil domain used in a crop include regenerated transformed plants, progeny of regenerated transformed plants, or plants produced by sexually crossing a transformed plant with a non-transformed plant.
One skilled in the art can easily make any necessary adjustments in accordance with the necessities of the particular situation.
Further objects and advantages of the present invention will be clear from the description that follows.